Developing Next‐Generation Protein‐Based Vaccines Using High‐Affinity Glycan Ligand‐Decorated Glyconanoparticles

Abstract Major diseases, such as cancer and COVID‐19, are frightening global health problems, and sustained action is necessary to develop vaccines. Here, for the first time, ethoxy acetalated dextran nanoparticles (Ace‐Dex‐NPs) are functionalized with 9‐N‐(4H‐thieno[3,2‐c]chromene‐2‐carbamoyl)‐Siaα2−3Galβ1−4GlcNAc (TCCSia‐LacNAc) targeting macrophages as a universal vaccine design platform. First, azide‐containing oxidized Ace‐Dex‐NPs are synthesized. After the NPs are conjugated with ovalbumin (OVA) and resiquimod (Rd), they are coupled to TCCSia‐LacNAc‐DBCO to produce TCCSia‐Ace‐Dex‐OVA‐Rd, which induce a potent, long‐lasting OVA‐specific cytotoxic T‐lymphocyte (CTL) response and high anti‐OVA IgG, providing mice with superior protection against tumors. Next, this strategy is exploited to develop vaccines against infection by severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). The receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein is the main target for neutralizing antibodies. The TCCSia‐Ace‐Dex platform is preferentially used for designing an RBD‐based vaccine. Strikingly, the synthetic TCCSia‐Ace‐Dex‐RBD‐Rd elicited potent RBD‐neutralizing antibodies against live SARS‐CoV‐2 infected Vero E6 cells. To develop a universal SARS‐CoV‐2 vaccine, the TCCSia‐Ace‐Dex‐N‐Rd vaccine carrying SARS‐CoV‐2 nucleocapsid protein (N) is also prepared, which is highly conserved among SARS‐CoV‐2 and its variants of concern (VOCs), including Omicron (BA.1 to BA.5); this vaccine can trigger strong N‐specific CTL responses against target cells infected with SARS‐CoV‐2 and its VOCs.


Materials S8
General methods for the synthesis S9

Synthesis of OVA FITC S17
Synthesis of TCC Sia-Ace-Dex-OVA FITC -Rd NPs S17 Synthesis of TCC Sia-Ace-Dex-OVA-Rd NPs S18 S3 Quantification of OVA, Rd and TCC Sia-LacNAc in the NPs S18 Table S1. The amounts of OVA/OVA FITC , TCC Sia-LacNAc, and Rd in the NPs. S18 The release profiles of OVA and Rd from the NPs S18 Preparation of CD169 + BMMs S19 Detection of OVA 257−264 presented by MHC-I of BMMs (CD169 + ) S19 B3Z T cell activation study S19 Immunization S19 In vivo CTL activity studies of OVA based NPs S20 Evaluation of antibody responses elicited by OVA based NPs S20

Antibody responses by TCC Sia-Ace-Dex-RBD-Rd NPs S21
Competitive ELISA S22 Table S3. The amounts of N protein, TCC Sia-LacNAc and Rd in the NPs. S23

Quantification of N 219−227 -MHC-I + CD8 + cells S23
In vivo CTL activity studies of N based NPs S23

Evaluation of antibody responses elicited by N based NPs S24
Product Characterization Spectra S25

References S33
S4 Figure S1. FTIR spectrum of Oxi-Ace-Dex-Az NPs (1). FTIR spectroscopy analysis revealed a signal at 1735 cm −1 in the Oxi-Dex-Az polymer and the Oxi-Ace-Dex-Az NPs (1) that was absent in the Dex and Dex-Az polymers without oxidation, indicating the presence of aldehyde groups on 1. Figure S2. In vivo toxicity of TCC Sia-Ace-Dex-OVA-Rd (17) by histological analysis.
C57BL/6 female mice were injected with EG7-OVA cells (1 × 10 6 ) on day 0. And a total of three subcutaneous immunizations with 17 were performed on days −7, 1, and 7. Then, mice were sacrificed by anesthesia on day 11. Organs from three mice immunized with 17 were harvested, sliced, and stained with hematoxylin/eosin (H&E). The results showed that no lesions were observed from these slides. Scale bars: 100 m. Figure S3. In vivo toxicity of TCC Sia-Ace-Dex-RBD-Rd (21)  induced higher levels of N-specific CTLs in vivo (see Figure 9), highlighting the importance of incorporating N protein into NP.    [2] . All processes in this study involving authentic SARS-CoV-2 were performed in a BSL-3 facility.

S9
Confocal microscopic images were performed on Zeiss LSM880 using the following filters: λex@488 nm and λem@500−530 nm for OVA FITC ; λex@543 nm and λem@555−600 nm for LysoTracker Red. In vivo CTL studies were performed on a BD Accuri™ C6 Plus Flow Cytometer. Transmission electron microscopy (TEM) images were acquired from FEI Tecnai G2 F20 (Thermo Fisher Scientific). The synthetic compounds were characterized by a high-resolution mass spectrometer (LCMS-IT-TOF, LC-20A) and nuclear magnetic resonance
The reaction solution was then dialyzed, during which time the water was changed 4−5 times.
The solution was lyophilized to obtain dextran-azide (Dex-Az

Synthesis of partially oxidized dextran-azide (Oxi-Dex-Az)
To dextran-azide (Dex-Az) (1 g) in water (10 mL) was added sodium periodate (0.22 g, 1.03 mmol). After stirring at RT for 5 h, the reaction mixture was dialyzed to remove small molecules. The product was lyophilized to give partially oxidized dextran-azide (Oxi-Dex-Az) as a white solid (0.7 g). Fourier transform infrared (FTIR) spectroscopy analysis shows a characteristic peak of the aldehyde group at 1735 cm −1 (see Figure S1).

Synthesis of Oxi-Ace-Dex-Az polymer
To the flask was added Oxi-Dex-Az polymer (1 g) and DMSO (10 mL). To the DMSO solution were added pyridinium p-toluenesulfonate (15.6 mg, 0.062 mmol) and S17 2-ethoxypropene (3.2 mL, 29.7 mmol) under nitrogen protection. After stirring at RT for 3 h, the reaction was quenched by adding triethylamine (1 mL). 100 mL of ultrapure water was then added. The mixture was centrifuged at 12,000 rpm for 20 min, and the pellet was washed with ultrapure water (50 mL × 2) and lyophilized to obtain the Oxi-Ace-Dex-Az

Synthesis of Oxi-Ace-Dex-Az NPs (1)
20 mg of Oxi-Ace-Dex-Az polymer was dissolved in DCM (1 mL). The solution was sonicated on ice for 1 min (4 sec on, 2 sec off) using a sonicator (Xinzhi JY96-IIN, with a duty cycle of 50%) to obtain a primary emulsion, which was added to 2 mL of H 2 O containing poly (vinyl alcohol) (PVA, Mw: 13−23 kg mol −1 , 3% w/w). After sonication on ice for 1 min, the emulsion was added to 0.3% (w/w) PVA in water (10 mL). After stirring for 3 h, the solvent was removed by centrifugation (12,000 rpm, 20 min). The particles were washed three times with ultrapure water (3 × 20 mL) and lyophilized to obtain Oxi-Ace-Dex-Az NPs, which were characterized by TEM and FTIR spectroscopy.
After stirring at 4 ℃ for 2 h, the reaction mixture was subjected to ultrafiltration (MWCO 30 kDa) to remove unconjugated FITC. Lyophilization of samples trapped in ultrafiltration tubes to obtain OVA FITC .

Synthesis of TCC Sia-Ace-Dex-OVA FITC -Rd NPs
To PBS (0.1M, pH 7.4, 200 L) containing Oxi-Ace-Dex-Az NPs (10 mg) was added OVA FITC (2 mg). The reaction was carried out at RT for 5 h. After that, resiquimod (Rd, 2 mg in 20 L DMSO) and TCC Sia-LacNAc-DBCO 2 (2 mg) were added to the solution, followed by stirring at RT overnight. The pellet was obtained by centrifugation (12000 rpm, 20 min) and washed 3 times with ultrapure water (3 × 20 mL). The resulting particles were then subjected to ultrafiltration against ultrapure water, followed by lyophilization to obtain TCC Sia-Ace-Dex-OVA FITC -Rd (11). For the synthesis of TCC Sia-Ace-Dex-OVA FITC (12), the synthetic steps were the same as those of 11, except that no Rd was added after the conjugation of Oxi-Ace-Dex-Az NPs with OVA FITC . In addition, PEG-Ace-Dex-OVA FITC -Rd S18 (13) and PEG-Ace-Dex-OVA FITC (14) were synthesized with reference to the synthetic steps of 11 and 12, respectively, except that the conjugated ligand 2 was replaced by DBCO-PEG 3 -OH (CAS: 2566404-76-8, 2 mg). Ace-Dex OVA FITC /Rd (15) and Ace-Dex OVA FITC (16) were prepared by the double-emulsion evaporation technique as described elsewhere. [4]

Quantification of OVA, Rd and TCC Sia-LacNAc in the NPs
The  (11) and TCC Sia-Ace-Dex-OVA-Rd (17), the NPs were degraded in water containing 0.5% TFA to release Rd. After ultrafiltration (MWCO 30 kDa), the absorbance at 345 nm of the intercepted precipitate was measured to quantify TCC Sia-LacNAc. In addition, the absorbance of the ultrafiltration filtrate at 321 nm was measured to quantify Rd.

The release profiles of OVA and Rd from the NPs
To test the release profiles of OVA and Rd, 0.5 mg of TCC Sia-Ace-Dex-OVA-Rd (17) and PEG-Ace-Dex-OVA-Rd (19) were respectively dissolved in 1 mL of 0.1 M PBS (pH 7. 4, 6.5, 6.0, 5.5 and 4.5) at 37 °C. At selected time intervals (6, 12, 24, 48 and 72 h), the supernatant was removed by centrifugation and the pellet was lyophilized. The amounts of unreleased OVA and Rd of the resulting particles were determined by the Bradford method and UV absorbance measurement, respectively.

Immunization
The priority immunization procedure for in vivo CTL detection was to immunize mice for a total of three injections weekly (Days 0, 7 and 14). To test the effect of dose or immunization interval on CTL induction, other immune procedures (see Figure 9g,h in the main text) were also tested. The immune process for humoral evaluation was to immunize mice or rabbits at two weeks intervals for three injections (Days 0, 14 and 28).

In vivo CTL activity studies of OVA based NPs
C57BL/6 mice were injected subcutaneously with free OVA, TCC Sia-Ace-Dex-OVA-Rd (17), TCC Sia-Ace-Dex-OVA (18), PEG-Ace-Dex-OVA-Rd (19) and PEG-Ace-Dex-OVA (20) via the neck on days 0, 7 and 14, respectively. All injected vaccines contained the same dose of OVA (100 g). Spleens were isolated from naive C57BL/6 mice on day 21 to prepare suspensions. Half of the splenocytes (2 × 10 7 , 2 mL) were labeled with a low concentration of carboxyfluorescein succinimidyl ester (CFSE concentration of 1 M), and placed in a CO 2 incubator for 10 min. By removing free CFSE from the cell mixture by centrifugation at 1600

EG7-OVA tumor challenge study
On day −7, C57BL/6 mice were immunized with PBS, free OVA, TCC Sia-Ace-Dex-OVA-Rd (17), and PEG-Ace-Dex-OVA-Rd (19), respectively. All injected vaccines contained the same dose of OVA (100 g). On day 0, EG7-OVA cells (1 × 10 6 ) were subcutaneously injected into the mice. Subsequently, two vaccine injections were given on the first and seventh days. Tumor volume was calculated with the formula: Volume (mm 3 ) = 1/2 (length × width × height). [6] Tumors were dissected on day 11, photographed, and weighed. The changes in the body weight of the mice were also monitored during this experiment.
IC 50 was calculated by fitting the inhibition from serially diluted serum to a sigmoidal dose-response curve. The positivity cut-off value was set at the mean of negative control (pre-immune sera) + 2 × SD (standard deviation). [7] Authentic SARS-CoV-2 virus neutralization test in Vero E6 cells Authentic virus neutralization titers were determined as previously described. [8] Briefly, serum antibodies were serially diluted in DMEM containing FBS (2.5%) and mixed with an equal volume of virus suspension. After 1 h of incubation, the mixture was added to 24-well plates containing Vero E6 monolayers for incubation of an additional 1 h. The inoculate was replaced with DMEM containing FBS (2.5%) and carboxymethylcellulose (0.9%). Plates were fixed with paraformaldehyde (8%) and stained with 0.5% crystal violet after 3 days.
Plaque reduction neutralizing titers were calculated using the "inhibitor versus normalized response (variable slope)" model in GraphPad (Prism 8.0) software. Cutoff values (geometric mean + 3 times geometric standard deviation) were calculated from negative controls.

Synthesis of TCC Sia-Ace-Dex-N-Rd (22) and TCC Sia-Ace-Dex-N 219−227 -Rd (23)
To 200 L of PBS (0.1 M, pH 7.4) containing 10 mg of Oxi-Ace-Dex-Az NPs (1) was added 2 mg of SARS-CoV-2 N protein (N) and 2 mg of Rd. After shaking at RT for 5 h, ligand 2 (2 mg) was added and stirred at RT overnight. After centrifugation (12000 rpm, 20 min), the pellet was washed with water (3 × 20 mL), followed by ultrafiltration to remove unbound N protein, Rd, and ligand 2. The resulting NPs were lyophilized to yield TCC Sia-Ace-Dex-N-Rd (22). In addition, TCC Sia-Ace-Dex-N 219−227 -Rd (23) was prepared similarly to 22, except that N 219−227 was used instead of the N protein. As a control, PEG-Ace-Dex-N-Rd (25) without TCC Sia-LacNAc was prepared in the same manner as 22, but DBCO-PEG 3 -OH was used instead of ligand 2 to conjugate with Oxi-Ace-Dex-Az NPs.  Spleens were isolated from naive C57BL/6 mice on day 14 to prepare suspensions. Half of the splenocytes (2 × 10 7 , 2 mL) were labeled with 1 M of CFSE and placed in a CO 2 incubator for 10 min. By removing free CFSE from the cell mixture by centrifugation at 1600 rpm for 3 min, CFSE lo N 219−227 − splenocytes were obtained. The other half of splenocytes (2 × 10 7 , 2 mL) were labeled with 10 M of CFSE, and then pulsed with N 219−227 with the sequence of LALLLLDRL (1 g·mL −1 ) for 1 h to obtain CFSE hi N 219−227 + splenocytes.

Evaluation of antibody responses elicited by N based NPs
On days 0, 14, and 28, C57BL/6 mice were immunized with free N, TCC Sia-Ace-Dex-N-Rd (22), PEG-Ace-Dex-N-Rd (25), and TCC Sia-Ace-Dex-Rd (24) + N, respectively. Sera were collected on days −1 (pre-immunization) and 35 for subsequent experiments. The procedure for antibody titer determination for N-based NPs is similar to that of OVA NPs. Different steps involved: SARS-CoV-2 N protein (N) (1 g N protein per S25 well) was coated on the plate. Second, the primary antibody-incubated serum was from mouse serum immunized with N-protein NPs. For rabbit immunization, on days 0, 14, and 28, rabbits (n = 3 per group) were vaccinated with free N, TCC Sia-Ace-Dex-N-Rd (22), PEG-Ace-Dex-N-Rd (25), respectively. Sera were collected on days −1, 21 and 35. To determine rabbit serum titers, ELISA was performed using HRP-conjugated goat anti-rabbit IgG.